Alcoholic Fermentation / Second Edition, 1914

One of the most interesting and at the same time most difficult problems concerning enzyme action in general is the nature of the inhibiting or accelerating effect produced by many substances upon the rate or total result of the chemical process set up in presence of the enzyme. Inhibition, it is usually supposed, involves either the decomposition of the enzyme, in which case it is irreversible, its removal from the sphere of action by some change in its mode of solution, or the formation of an inactive or less active compound between the enzyme and the inhibiting agent. This compound it may sometimes be possible to decompose, with the result that the activity of the enzyme is restored. A striking example of this, to which allusion has already been made, is the effect of hydrocyanic acid on alcoholic fermentation (p. 37).

Acceleration of enzyme action can in some cases be ascribed to the fact that the accelerating substance possesses an assignable chemical function in the reaction, so that an increase in the concentration of this substance causes an increase in the rate of the reaction. As we have seen in Chapter III, this is the explanation of the accelerating effect of phosphates on fermentation by yeast-juice. In many other cases, however, no such chemical function can be traced, as, for example, in the effect of neutral salts on the hydrolytic action of invertase, or the effect of the addition of the co-enzyme to zymase, and it is necessary to fall back on some assumption, such as that the accelerating agent acts by increasing the effective concentration of the enzyme or by combining either with the enzyme or the substrate, forming a compound which undergoes the reaction more readily.

The interest in the following examples of inhibition and acceleration of fermentation by yeast-juice lies not only in their relation to these general problems but also, and perhaps chiefly, in their bearing on the specific problem of the nature and mode of action of the various agents concerned in the production of alcohol and carbon dioxide from sugar in the yeast-cell. [p071]

As the concentration of phosphate is still further increased, it is first observed that the maximum velocity, which is still attained almost immediately after the addition of the phosphate, is maintained for a certain period before the fall commences, and then, as the increase in concentration of phosphate proceeds, that the maximum is only gradually attained after the addition, the period required for this increasing with the concentration of the phosphate. Moreover, with still higher concentrations, the maximum rate attained is less than that reached with lower concentrations, and further, the rate falls off more slowly. The concentration of phosphate which produces the highest rate, which may be termed the optimum concentration, varies very considerably with different specimens of yeast-juice [Harden and Young, 1908, 1].

All these points are illustrated by the accompanying curves (Fig. 7) which show the rate of evolution per five minutes plotted against the time for four solutions in which the initial concentrations of phosphate were (A) 0·033, (B) 0·067, (C) 0·1, and (D) 0·133 molar, the volumes of 0·3 molar phosphate being 5, 10, 15, and 20 c.c. in each case added to 25 c.c. of yeast-juice, and made up to 45 c.c, each solution containing 4·5 grams of glucose. The time of addition is taken as zero, the rate before addition being constant, as shown in the curves.

It will be observed that 5 and 10 c.c. (A and B) give the same maximum, whilst 15 c.c. (C) produce a much lower maximum, and 20 c.c. (D) a still lower one, the rate at which the velocity diminishes after the attainment of the maximum being correspondingly slow in these last two cases. By calculating the amount of phosphate which has disappeared as such from the amount of carbon dioxide evolved, [p072] it is found that the maximum does not occur at the same concentration of free phosphate in each case.

It is also possible as suggested by Buchner for the analogous case of arsenite (p. 78) that the addition of increasing amounts of phosphate causes a progressive but reversible change in the mode of dispersion [p073] of the colloidal enzyme, and that this has the secondary effect of altering the rate of fermentation. No decisive evidence is as yet available upon the subject.

The results obtained by Euler and Johansson [1913] to which reference has already been made indicate that in presence of a moderate excess of phosphate esterification is more rapid than production of carbon dioxide. No explanation of this phenomenon has yet been given, but it might obviously be due either to the production of some phosphorus compound which subsequently takes part in the production both of hexosediphosphate and of carbon dioxide, or, less probably, to the entire independence of the two changes—esterification of phosphate and production of carbon dioxide—which might then be differently affected by the presence of excess of phosphate and therefore take place at different rates.

II. Reaction of Fructose with Phosphates in Presence of Yeast-Juice.

Although, as has been pointed out (p. 42), glucose, mannose, and fructose all react with phosphate in a similar manner in presence of yeast-juice, there are nevertheless certain quantitative differences between the behaviour of glucose and mannose on the one hand, and fructose on the other, which appear to be of considerable importance. Fructose differs from the other two fermentable hexoses in two particulars: (1) the optimum concentration of phosphate is much greater; (2) the maximum rate of fermentation attainable is much higher [Harden and Young, 1908, 2; 1909].

These points are clearly illustrated by the following results, which all refer to 10 c.c. of yeast-juice, and show that the optimum concentration of phosphate for the fermentation of fructose is from 1·5 to 10 times that of glucose, and that the maximum rate of fermentation for fructose in presence of phosphate is 2 to 6 times that of glucose.

Another way of expressing the same thing is to say that the optimum concentration of phosphate (p. 71) is greatly raised when 2·5 per cent. of fructose is added to glucose, and that consequently the rate of fermentation rises. The effect is extremely striking, since a mixture of glucose and yeast-juice fermenting in the presence of a large excess of phosphate at the rate of less than 1 c.c. of carbon dioxide in five minutes may be made to ferment at six to eight times this rate by the addition of only 0·05 gram of fructose (2·5 per cent. of the glucose present), and to continue until the total gas evolved is at least five to six times as great as that obtainable from the added fructose, the concentration of the phosphate being the whole time at such a height as would limit the fermentation of glucose alone to its original value.

The effect is not produced when the concentration of the phosphate is so high that the rate of fermentation of fructose is itself greatly lowered.

This remarkable inductive effect is specific to fructose and is not produced when glucose is added to mannose or fructose, or by mannose when added to glucose or fructose, under the proper conditions of concentration of phosphate in each case.

This interesting property of fructose, taken in connection with the [p075] facts that this sugar in presence of phosphate is much more rapidly fermented than glucose or mannose, and that the optimum concentration of phosphate for fructose is much higher than for glucose or mannose, appears to indicate that fructose when added to yeast-juice does not merely act as a substance to be fermented, but in addition, bears some specific relation to the fermenting complex.

The close analogy which exists between the chemical functions of phosphorus and arsenic lends some interest to the examination of the action of sodium arsenate upon a mixture of yeast-juice and sugar, and experiments reveal the fact that arsenates produce a very considerable acceleration in the rate of fermentation of such a mixture [Harden and Young, 1906, 3; 1911, 1]. The phenomena observed, however, differ markedly from those which accompany the action of phosphate.

The acceleration produced is of the same order of magnitude as that obtained with phosphate, but it is maintained without alteration for a considerable period, so that there is no equivalence between the amount of arsenate added and the extra amount of fermentation effected. Further, no organic arsenic compound corresponding in composition with the hexosephosphates appears to be formed.

Increase of concentration of arsenate produces a rapid inhibition of [p076] fermentation, probably due to some secondary effect on the fermenting complex, possibly to be interpreted as the formation of compounds incapable of combining with sugar and hence unable to carry on the process of fermentation. An optimum concentration of arsenate therefore exists just as of phosphate, at which the maximum rate is observed, and this optimum concentration and the corresponding rate vary with different samples of juice and are less for glucose than for fructose. The rate of fermentation by zymin is relatively less increased than that by yeast-juice.

The contrast between glucose and fructose in their relations to [p077] arsenate are well exhibited in the following table, in which the rates of fermentation produced by arsenate in presence of excess of glucose and fructose respectively are given:—

Effects somewhat similar to those produced by arsenates were observed by Buchner [Buchner and Rapp, 1897; 1898, 1, 2, 3; 1899, 2; Buchner, E. and H., and Hahn, 1903, pp. 184–205] when potassium arsenite was added to yeast-juice. This substance, the action of which on yeast had been adduced by Schwann as a proof of the vegetable nature of this organism, was employed by Buchner on account of its poisonous effect on vegetable cells as an antiseptic and as a means of testing for the protoplasmic nature of the agent present in yeast-juice. Its effect on the fermentation was, however, found to be irregular, and at the same time it did not act as an efficient antiseptic in the concentrations which could be employed. Even 2 per cent. of arsenious oxide, added as the potassium salt, had in many cases a decided effect in diminishing the total fermentation obtained with cane sugar, and this effect increased with the concentration. A number of irregularities were also observed which cannot here be discussed. It was further found that in some cases 2 per cent. of arsenious oxide inhibited the fermentation of glucose but not of saccharose, or of a mixture of glucose and fructose, whilst its effect on fructose alone was of an intermediate character.

The important observation was also made by Buchner that the addition of a suitable quantity of arsenite as a rule caused a greatly increased fermentation during the first sixteen hours even in experiments in which the total fermentation was diminished. By examining the effect of arsenite on fermentation in a similar manner to that of arsenate, Harden and Young [1911, 1] have found that a close analogy exists [p078] between the effects and modes of action of these substances, but that arsenite produces a much smaller acceleration than arsenate. An optimum concentration of arsenite exists, just as in the case of arsenate, which produces a maximum rate of fermentation. Further increase in concentration leads to inhibition, and in no case is there any indication of the production of an exactly equivalent amount of fermentation as in the case of phosphate. In various experiments with dialysed, evaporated, and diluted yeast-juice in which 2 per cent. of arsenious oxide was found by Buchner to inhibit fermentation, it is probable that, owing to the small amount of fermenting complex left, this amount of arsenious oxide was considerably in excess of the optimum concentration, although Buchner ascribes the effect to the removal of some of the protective colloids of the juice, owing to the prolonged treatment to which it had been subjected.

The inhibiting effects of higher concentrations of arsenite and arsenate also present close analogies, but this most interesting aspect of the question has not yet been sufficiently examined to repay detailed discussion. Buchner [Buchner, E. and H., and Hahn, 1903, pp. 199–205] has suggested that the inhibition is due primarily to some change in the colloidal condition of the enzyme and has shown that certain colloidal substances appear to protect it, as does also sugar. The possibility is also present that inactive combinations of some sort are formed between the fermenting complex and the inhibiting agent, in the manner suggested to account for the inhibiting effect of excess of phosphate (p. 72). It seems most probable that the effect is a complex one, in which many factors participate.

Nature of the Acceleration Produced by Arsenate and Arsenite.

In explanation of the remarkable accelerating action of arsenates and arsenites two obvious possibilities present themselves. In the [p079] first place the arsenic compound may actually replace phosphate in the reaction characteristic of alcoholic fermentation, the resulting arsenic analogue of the hexosephosphate being so unstable that it undergoes immediate hydrolysis, and is therefore only present in extremely small concentration at any period of the fermentation and cannot be isolated. In the second place it is possible that the arsenic compound may accelerate the action of the hexosephosphatase of the juice, and thus by increasing the rate of circulation of the phosphate produce the permanent rise of rate. With this effect may possibly be associated a direct acceleration of the action of the fermenting complex.

The experimental decision between these alternative explanations is rendered possible by the use of a mixture of enzyme and co-enzyme free from phosphate and hexosephosphate. As has already been described (p. 55) a mixture of boiled yeast-juice, which has been treated with lead acetate, glucose or fructose, and washed zymin can be prepared which scarcely undergoes any fermentation unless phosphate be added. If now arsenates or arsenites can replace phosphate, they should be capable of setting up fermentation in such a mixture. Experiment shows that they do not possess this power. For fermentation to proceed phosphate must be present and it cannot be replaced either by arsenate or arsenite [Harden and Young, 1911, 1].

The effect of these salts on the action of the hexosephosphatase can also be ascertained by a modification of the foregoing experiment. If a hexosephosphate be made the sole source of phosphate in such a mixture as that described above, in which it must be remembered abundance of sugar is present, the rate at which fermentation can proceed will be controlled by the rate at which the hexosephosphate is decomposed with formation of phosphate. Experiment shows that in the presence of added arsenate or arsenite the rate of fermentation is largely increased, so that the effect of these salts must be to increase the rate of liberation of phosphate, or in other words, to accelerate the hydrolytic action of the hexosephosphatase.

This conclusion is even more strikingly confirmed by a comparison of the direct action of yeast-juice on hexosephosphate in presence and in absence of arsenate, as measured by the actual production of free phosphate. In a particular experiment this gave rise to 0·0707 gram of Mg₂P₂O₇ in the absence of arsenate and 0·6136 gram of Mg₂P₂O₇ in the presence of arsenate.

The results obtained with arsenite are throughout very similar to those given by arsenate, but are not quite so striking. It may therefore be affirmed with some confidence that the chief action of arsenates [p080] and arsenites in accelerating the rate of fermentation of sugars by yeast-juice or zymin, consists in an acceleration of the rate at which phosphate is produced from the hexosephosphate by the action of the hexosephosphatase.

CHAPTER VI. CARBOXYLASE.

It was found that yeast had the power of rapidly decomposing a large number of hydroxy-and keto-acids [Neuberg and Hildesheimer, 1911; Neuberg and Tir, 1911; see also Karczag, 1912, 1, 2]. The most important among these are pyruvic acid, CH₃·CO·COOH, and a considerable number of other aliphatic a-keto-acids which are decomposed with evolution of carbon dioxide and formation of the corresponding aldehyde:—

The reaction is produced by all races of brewer's yeast which have been tried, as well as by active yeast preparations and extracts and by wine yeasts [Neuberg and Karczag, 1911, 4; Neuberg and Kerb, 1912, 2]. The phenomenon can readily be exhibited as a lecture experiment by shaking up 2 g. of pressed yeast with 12 c.c. of 1 per cent. pyruvic acid, placing the mixture in a Schrötter's fermentation tube, closing the open limb by means of a rubber stopper carrying a long glass tube and plunging the whole in water of 38–40°. Comparison tubes of yeast and water and yeast and 1 per cent. glucose may be started at the same time, and it is then seen that glucose and pyruvic acid are fermented at approximately the same rate [Neuberg and Karczag, 1911, 1]. If English top yeast be used it is well to take 0·5 per cent. pyruvic acid solution and to saturate the liquids with carbon dioxide before commencing the experiment. The production of acetaldehyde can be readily demonstrated by distilling the mixture at the close of fermentation and testing for the aldehyde either by Rimini's reaction (a blue coloration with diethylamine and sodium nitroprusside) or by means of p-nitrophenylhydrazine which precipitates the hydrazone, melting at 128·5° [Neuberg and Karczag, 1911, 2, 3]. [p082]

Under these conditions a considerable portion of the aldehyde undergoes condensation to aldol [Neuberg, 1912]:—

This change appears to be due entirely to the alkali and not to an enzyme since the aldol obtained yields inactive β-hydroxybutyric acid on oxidation [Neuberg and Karczag, 1911, 3; Neuberg, 1912]. The various preparations derived from yeast which are capable of producing alcoholic fermentation also effect the decomposition of pyruvic acid in the same manner as living yeast. They are, however, more sensitive to the acidity of the pyruvic acid, and it is therefore advisable to employ a salt of the acid in presence of excess of a weak acid, such as boric or arsenious acid, which decomposes the carbonate formed but has no inhibiting action on the enzyme [Harden, 1913; Neuberg and Rosenthal, 1913].

As already mentioned the action is exerted on α-ketonic acids as a class and proceeds with great readiness with oxalacetic acid, COOH·CH₂·CO·COOH, all the three forms of which are decomposed, with α-ketoglutaric acid, and with α-ketobutyric acid. Hydroxypyruvic acid CH₂(OH)·CO·COOH is slowly decomposed yielding glycolaldehyde, CH₂(OH)·CHO, and this condenses to a sugar [Neuberg and Kerb, 1912, 3; 1913, 1]. Positive results have also been obtained with diketobutyric, phenylpyruvic, p-hydroxyphenylpyruvic, phenylglyoxylic and acetonedicarboxylic acids [Neuberg and Karczag, 1911, 5].

Relation of Carboxylase to Alcoholic Fermentation.

With regard to the relation of carboxylase to the process of alcoholic fermentation, nothing definite is yet known. As Neuberg points out [see Neuberg and Kerb, 1913, 1] the universal presence of the enzyme in yeasts capable of producing alcoholic fermentation, and the extreme readiness with which the fermentation of pyruvic acid takes place create a [p083] strong presumption that the decomposition of pyruvic acid actually forms a stage in the process of the alcoholic fermentation of the sugars. On the other hand Ehrlich's alcoholic fermentation of the amino-acids (p. 87) provides another function for carboxylase—that of decomposing the α-ketonic acids produced by the deaminisation of the amino-acids. It must be remembered in this connection that carboxylase is not specific in its action, but catalyses the decomposition not only of pyruvic acid but also of a large number of other α-ketonic acids, including many of those which correspond to the amino-acids of proteins and are doubtless formed in the characteristic decomposition of these amino-acids by yeast. Carboxylase undoubtedly effects one stage in the production of alcohols from amino-acids, whether it is also the agent by which one stage in the alcoholic fermentation of sugar is brought about still remains to be proved.

A comparison of the conditions of action of carboxylase and zymase has revealed several interesting points of difference. Neuberg and Rosenthal [1913] have observed that the fermentation of pyruvic acid by maceration extract commences much more rapidly than that of glucose and interpret this to mean that in the fermentation of glucose a long preliminary process occurs before sufficient pyruvic acid has been produced to yield a perceptible amount of carbon dioxide. The long delay (3 hours) which they sometimes observed in the action of maceration juice on glucose is however by no means invariable (see p. 46), but in any case indicates that the sugar fermentation can be affected by conditions which are without influence on the pyruvic fermentation. A similar conclusion is to be drawn from the fact that the pyruvic acid fermentation is less affected by antiseptics than the glucose fermentation [Neuberg and Karczag, 1911, 4; Neuberg and Rosenthal, 1913], chloroform sufficient to stop the glucose fermentation brought about by yeast or dried yeast being usually without effect on the fermentation of the pyruvates either alone or in presence of boric or arsenious acid. A more important difference is that carboxylase decomposes pyruvic acid in the absence of the co-enzyme which is necessary for the fermentation of glucose [Harden, 1913; Neuberg and Rosenthal, 1913]. This can be demonstrated experimentally by washing dried yeast or zymin with water (see p. 63) until it is no longer capable of decomposing glucose (Harden), or by allowing maceration extract to autolyse or dialyse until it is free from co-enzyme (Neuberg and Rosenthal). The zymase of maceration extract is moreover inactivated in 10 minutes at 50–51°, whereas after this treatment the carboxylase is still active. [p084]

CHAPTER VII. THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION.

All forms of alcohol prepared by fermentation contain a fraction of high boiling-point, which is termed fusel oil, and amounts to about [p086] 0·1 to 0·7 per cent. of the crude spirit obtained by distillation. This material is not an individual substance, but consists of a mixture of very varied compounds, all occurring in small amount relatively to the ethyl alcohol from which they have been separated. The chief constituents of the mixture are the two amyl alcohols, isoamyl alcohol,

The origin of these purely non-nitrogenous compounds was usually sought in the sugar of the liquid fermented, from which they were thought to be formed by the yeast itself or by the agency of bacteria [Emmerling, 1904, 1905; Pringsheim, 1905, 1907, 1908, 1909], whilst others traced their formation to the direct reduction of fatty acids. Felix Ehrlich has, however, conclusively shown in a series of masterly researches that the alcohols, and probably also the aldehydes, contained in fusel oil are in reality derived from the amino-acids which are formed by the hydrolysis of the proteins.

The close relationship between the composition of leucine,

and isoamyl alcohol,

had previously led to the surmise that a genetic relation might exist between these substances, but the idea had not been experimentally confirmed. In 1903 Ehrlich discovered [1903; 1904, 1, 2; 1907, 2; 1908; Ehrlich and Wendel, 1908, 2] that proteins also yield on hydrolysis an isomeride of leucine known as isoleucine, which has the constitution